Abstract

We report a new image processing technique for the structured illumination microscopy designed to work with low signals, with the goal of reducing photobleaching and phototoxicity of the sample. Using a pre-filtering process to estimate experimental parameters and total variation as a constraint to reconstruct, we obtain two orders of magnitude of exposure reduction while maintaining the resolution improvement and image quality compared to a standard structured illumination microscopy. The algorithm is validated on both fixed and live cell data with results confirming that we can image more than 15x more time points compared to the standard technique.

Figures (8)

Central bands of beads data with high and low signal levels indicated by “H” or “L” inside the parenthesis. (Left column: high exposure with transmission filter set at 10%; Right: low exposure with transmission filter set at 0.1%. The exposure time for both cases is 5 ms). DL: diffraction limited image (a–b); 0th band (the central band) is the Fourier transform of the average of 5 phase-shifted images, the amplitudes of which at the fz = 0 plane are shown in (c–d). Cross-sections of 0th bands (e–f): the vertical lines mark the lateral cutoff frequency of the objective; solid line (blue online) is averaged over the fx and fy axes while the dashed line (red online) is averaged over other areas at the fz = 0 plane.

Actin results with high and low signal levels indicated by “H” or “L” inside the parenthesis (Transmission filter is 30% for high signal and 1% for low signal; exposure times are 10 ms for both cases.) Wiener: reconstructed by the standard Wiener filtering where experimental parameters are estimated without pre-filtering; WP: Wiener method with pre-filtering. Scale bar: 5 μm.

Intensity decay with high and low exposure experiments whose results are shown in Fig. 6 and 7. Dashed line (blue online) is the power law curve for the high signal case and the solid line (blue online) is the power law curve for the low signal case.